Prolonged release biodegradable microspheres and method for preparing same

ABSTRACT

The invention concerns a method for preparing a pharmaceutical composition in the form of microspheres with prolonged release of a water-soluble active principle. The invention is characterized in that it comprises the following successive steps: dissolving an active principle in an appropriate amount of water; emulsifying the resulting aqueous solution of active principle with a d,l-lactid-co-glycolide matrix copolymer, of average molecular weight ranging between 40000 and 80000, dissolved in a chlorinated hydrocarbon, resulting in a first microfine and homogeneous emulsion; emulsifying said resulting first emulsion in an external aqueous phase, containing a surfactant, a viscosity-enhancing agent and an osmotic agent; extraction-evaporation of the solvent to obtain microspheres which are recuperated after filtering, washing and drying. The invention also concerns microspheres obtainable by implementing said method, having continuous release over a period of more than two months, advantageously over a period of at least three months.

The present invention relates to a method for preparing a pharmaceuticalcomposition in the form of microspheres with prolonged release of awater-soluble active principle. The invention also relates tomicrospheres which can be obtained using this method, and which exhibitcontinuous release of active principle over a period of more than twomonths, advantageously of at least three months.

Many pharmaceutical compositions, in the form of biodegradable polymer-and copolymer-based microspheres, containing pharmacologically activecompounds and designed for controlled and prolonged release of saidcompounds, have been described in various documents of the prior stateof the art. Such pharmaceutical compositions have great value for thetreatment of various diseases requiring continuous and prolonged releaseof active principle.

However, the formulations developed to date for setting up this type ofcomposition have various disadvantages and few of them reach the stageof clinical trials.

One of the major problems associated with these prolonged releaseformulations is the release of a large amount of active principle duringthe first hours following administration of the pharmaceuticalcomposition. “Burst” effect or “burst” release are terms commonly usedin reference to such a release. This release generally results in anabrupt increase in plasma concentrations of the medicinal product,which, in many cases, results in toxicological problems which areunacceptable for humans. This “burst” release also results in a decreasein the duration of activity of the pharmaceutical composition due to theabrupt and rapid release of a large amount of said active principlesubsequent to the administration of the composition.

A second problem lies in the fact that relatively inefficientencapsulation rates are generally obtained using the conventionalmethods of microencapsulation, in particular when the active principleis a water-soluble medicinal product.

A third problem which must be solved in developing these formulations isthe instability of the active principles in the face of the rigorousconditions used in producing the microspheres, such as high temperaturesor prolonged contact of the active principle with organic solventsduring the solvent evaporation step.

Several trials have been carried out in order to solve these variousproblems. Thus, additives such as sugars, oils, wax, proteins, polymers,salts or acids have been used in the preparation of pharmaceuticalcompositions in the form of microspheres. These additives, which act assubstances for retaining the medicinal product in the microsphere, makeit possible to increase the efficiency of the method ofmicroencapsulation and even, possibly, to protect the active principleduring the process, by playing the role of stabilizing agents.

However, the inclusion of these additives in the microspheres can leadto problems of interaction between the additives and the activeprinciple or the polymer-based matrix, thus inducing problems in termsof toxicology and of pharmacological activity of the medicinal product.In addition, the additives, which retain the active principle inside themicrospheres during the production process, have an influence on therelease profile of the active principle contained in the microspheres,possibly preventing continuous release of said active principlesubsequent to administration of the microspheres.

Other methods of microencapsulation have also been developed in anattempt to increase the efficiency of microencapsulation of the activeprinciple within the microspheres, based on the use of mixtures oforganic solvents, but such methods lead to problems of stability of theactive principle during the microsphere production process.

Consequently, there was a need to develop a method for preparing apharmaceutical composition in the form of biodegradable polymer- andcopolymer-based microspheres designed for prolonged release of awater-soluble active principle, which does not have the disadvantages ofthe compositions described in the documents of the prior state of theart or of the compositions developed to date.

The present invention satisfies this need. The applicant has thusdiscovered, surprisingly, that the development of microspheres based onactive principle and on biodegradable d,l-lactide-co-glycolide matrixcopolymer having a specific molecular weight and a specific lacticacid/glycolic acid ratio, using a rapid multiple emulsion/solventevaporation method, without using any additive or a modulating agent asis the case in patent FR 2 718 642, makes it possible to obtainmicrospheres exhibiting continuous and sustained release of activeprinciple over a period of more than two months, while at the same timeexhibiting a limited “burst” effect. Such a method of encapsulation,which uses conditions which are gentle and relatively nonaggressive forthe medicinal product, also makes it possible to preserve the stabilityof said medicinal product and to obtain a homogeneous distribution ofthe medicinal product within the microsphere obtained.

The physical principle of multiple emulsion for encapsulatingwater-soluble active principles has in particular been described in U.S.Pat. No. 3,523,906. In general, in a method of this type by W/O/Wmultiple emulsion and solvent evaporation, the water-soluble activeprinciple is first of all solubilized in the internal phase of a firstW/O emulsion, and then, secondly, this first emulsion is in turnemulsified in an external aqueous phase.

The applicant has discovered, surprisingly, that the use of an osmoticagent during the step for emulsifying the first emulsion in the externalaqueous phase makes it possible to obtain a particularly highencapsulation efficiency by increasing the content of active principleencapsulated within the polymer matrix, and to influence the size of themicrospheres.

The subject of the present invention, relating to a rapid, veryefficient method of encapsulation which is relatively nonaggressive forthe active principle, thus makes it possible to obtain microspheres,based on water-soluble active principle and on d,l-lactide-co-glycolidematrix copolymer, exhibiting continuous and sustained release of activeprinciple over a period of more than two months, preferably of at leastthree months, while at the same time exhibiting a small “burst” effectduring the hours following administration of the composition.

A subject of the present invention is thus a method for preparing apharmaceutical composition in the form of microspheres with prolongedrelease of a water-soluble active principle, characterized in that itcomprises the succession of steps below:

-   -   dissolving the active principle in an appropriate amount of        water,    -   emulsifying the aqueous solution of active principle thus        obtained with a solution of a d,l-lactide-co-glycolide matrix        copolymer of average molecular weight of between 40 000 and 80        000 and having a lactic acid/glycolic acid proportion of between        50/50 and 80/20, dissolved in a chlorinated hydrocarbon,        resulting in a first microfine and homogeneous emulsion, the        size of said emulsion being advantageously less than 1 μm,    -   emulsifying said first emulsion thus obtained, in an external        aqueous phase containing a surfactant, a viscosity-increasing        agent and an osmotic agent,    -   extracting-evaporating the solvent so as to obtain microspheres,        which are recovered after filtration, washing and drying.

Other characteristics and advantages will emerge on reading the detaileddescription given below, in particular based on some examples ofparticular implementations.

The water-soluble active principle which can be used in the context ofthe method according to the present invention is advantageously chosenfrom the group consisting of peptides, proteins, vaccines, antibiotics,antidepressants, analgesics, anti-inflammatories and cytostatics. Evenmore advantageously according to the present invention, the activeprinciple is 5-OxoPro-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-ProNHEt or one ofits salts. This active principle is a GnRH hormone analogue with agonistactivity.

In the context of the method according to the present invention, thefirst step for dissolving the active principle in water so as to form aninternal aqueous phase is carried out without the addition of anysubstance which retains the active principle, nor of agent forstabilizing the emulsion, and without any operation intended to increasethe viscosity. Advantageously according to the present invention, theactive principle is dissolved in the internal aqueous phase without anyadditive or adjuvant.

The concentrations used in the internal aqueous phase in the context ofthe present invention depend on the water-solubility of the activeprinciple, on the characteristics of said active principle and on thedesired duration of release to be obtained. Advantageously according tothe present invention, the active principle is present at aconcentration of between 0.01 and 95% by weight, advantageously between0.5 and 40% by weight, relative to the total weight of the internalaqueous phase.

The first emulsion can in particular be formed using an ultrasounddevice or a homogenizer.

The matrix copolymer which can be used, according to the presentinvention, for preparing microspheres by W/O/W must be able to besolubilized in an appropriate volatile solvent, such as halogenatedalkanes. Advantageously according to the present invention, the solventis a chlorinated hydrocarbon such as methylene chloride, chloroform,chloroethane, dichloroethane or trichloroethane. Even moreadvantageously according to the present invention, the chlorinatedhydrocarbon is methylene chloride.

The d,l-lactide-co-glycolide matrix copolymer which can be used in themethod according to the present invention has the cumulative advantagesof being water-insoluble, of being biodegradable (such a copolymer isabsorbed without accumulating in the vital organs and is, finally,completely eliminated), of being biocompatible with the organism and ofbeing perfectly tolerated by it, and, finally, of having a minimalinflammatory response.

In the context of the method according to the present invention, thesolution of the d,l-lactide-co-glycolide matrix copolymer is obtained bydissolving the copolymer in a chlorinated hydrocarbon such as methylenechloride, without the addition of any release-modulating agent. In fact,it has been discovered that the use of a release-modulating agent is notsuitable for the production of microspheres designed for release over aperiod of time of more than two months.

The selection made with respect to the copolymer, with a specificmolecular weight and a specific lactic acid/glycolic acid ratio,combined with the fact of not using a release-modulating agent in themethod which is the subject of the present invention thus provides theadvantage of obtaining microspheres exhibiting continuous and sustainedrelease of active principle over a period of more than two months,preferably of at least three months, while at the same time exhibiting alimited “burst” effect during the hours following administration of thecomposition.

The concentrations of the polymer dissolved in the organic solution ofmethylene chloride depend on the active principle and on the desiredrate of release. Advantageously according to the method which is thesubject of the present invention, the matrix copolymer is present at aconcentration of between 5 and 50% by weight relative to the totalweight of the solution consisting of the copolymer dissolved in thechlorinated hydrocarbon.

According to the method which is the subject of the present invention,the external aqueous phase contains a surfactant such as polysorbate 80,a viscosity-increasing agent such as polyvinylpyrrolidone and an osmoticagent such as mannitol or sodium chloride. Advantageously according tothe present invention, the external aqueous phase contains a solution ofpolysorbate 80, of polyvinylpyrrolidone and of mannitol or of sodiumchloride. Even more advantageously according to the present invention,the external aqueous phase contains a solution of polysorbate 80, ofpolyvinylpyrrolidone and of sodium chloride.

Advantageously according to the method which is the subject of thepresent invention, the surfactant is present at a concentration ofbetween 0.1 and 0.5% by weight, the viscosity-increasing agent ispresent at a concentration of between 1 and 25% by weight and theosmotic agent is present at a concentration of between 0.1 and 10% byweight, relative to the total weight of the external aqueous phase. Thecomposition of the external aqueous phase is determinant for theformation of the second emulsion (emulsification of the first emulsion),and it is, consequently, determinant for the production of themicrospheres. It thus contributes to the rapid stabilization of themicrospheres, it influences the efficiency of encapsulation of theactive principles, and it constitutes an essential factor forcontrolling the size of the final microspheres and their morphology.

During the step consisting of extraction-evaporation of the solvent, thesolvent is rapidly eliminated at ambient temperature and underatmospheric pressure, according to a continuous evaporating systemconsisting of a slope of adequate length, over which the suspension ofmicrospheres flows in a thin layer.

Such a system for continuous evaporation of the organic solvent, whichincreases and promotes contact between the emulsion and the air, makesit possible to obtain rapid stabilization of the polymer matrix, theeffect of which is to increase the stabilization of the active principlewhile at the same time trapping a high percentage of said principlewithin the microspheres (efficiency of encapsulation), and to producerapid evaporation of the solvent, the effect of which is to reduce thetotal time for implementation of the method. The rapid stabilization ofthe polymer matrix obtained during this step for extraction-evaporationof the organic solvent also makes it possible to obtain a homogeneousdistribution of the active substance throughout the microsphere (thusmaking it possible to obtain a low concentration of active principleencapsulated close to the surface of the microsphere), thus contributingto decreasing the phenomenon of “burst” release, subsequent toadministration of the microspheres. In addition, implementing the methodat ambient temperature and under atmospheric pressure makes it possibleto avoid problems such as alteration of thermolabile products or ruptureof the microspheres when a vacuum is used during theextraction-evaporation step.

In a more detailed manner, the method for producing the microspheresaccording to the present invention comprises the following steps:

A certain amount of active principle is dissolved in a volume of water.This solution is emulsified, using an ultrasound device for example, ina volume of methylene chloride containing a poly(lactide-co-glycolide)copolymer of average molecular weight of between 40 000 and 80 000daltons and having a proportion of lactic acid to glycolic acid ofbetween 50/50 and 80/20. The first emulsion which results therefromshould be microfine and homogeneous, thus making it possible to spreadthe active principle throughout the polymer matrix, ensuringreproducibility of the various batches, without needing to usesurfactants or other adjuvants. Once the first emulsion is formed, it isin turn emulsified in an external phase, consisting of an aqueoussolution of polysorbate 80 as surfactant, of polyvinylpyrrolidone asviscosity-increasing agent and of mannitol or NaCl as osmotic agent,with stirring for a short period of time. After this step, the doubleemulsion is diluted with water, and the suspension is then passed, underatmospheric conditions, over a continuous evaporating system consistingof a slope of adequate length, over which the suspension of microspheresflows in a thin layer. This system makes it possible to promote contactbetween the emulsion and the atmosphere, which reduces the amount oftime for evaporation of the organic solvent, and to increase thestabilization of the active principle. The microspheres obtained in thisway are then recovered by filtration, washed with water, and dried bylyophilization.

The microspheres obtained according to the method which is the subjectof the present invention are microspheres with a high content of activeprinciple, allowing continuous release in vivo of the medicinal productover a period of more than two months, preferably of at least threemonths, with a small “burst” effect, after parenteral administration ofthe microspheres.

A subject of the present invention is also the microspheres which can beobtained by implementing the method according to the present invention,exhibiting continuous release of active principle over a period of morethan two months, advantageously over a period of at least thee months.

Advantageously according to the present invention, the active principleis present at a concentration of between 0.5 and 20% by weight,advantageously between 5 and 15% by weight, relative to the total weightof the microspheres.

Advantageously according to the present invention, the microspheres areless than 250 μm, even more advantageously less than 90 μm, in size.This size is suitable for administration of the microspheres.

The microspheres according to the present invention are advantageouslyadministered parenterally, even more advantageously in the form ofintramuscular, subcutaneous or intra-arterial injections, or injectionsat the site of a tumor. For suitable administration, the microspheresare preferably dispersed in a standard aqueous medium with dispersingagents and isotonic agents.

The following examples (studies in vivo and in vitro) are given in anonlimiting capacity and illustrate the present invention.

EXAMPLE 1 (COMPARATIVE)

Batches of microspheres based on5-OxoPro-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-ProNHEt acetate are prepared inaccordance with the method of production described in the publicationAdvanced Drug Delivery Reviews, 2.8 (1997), 43-70, which differs fromthe method which is the subject of the present invention. Themicrospheres are produced by dissolving an appropriate amount of activeprinciple in water, and then by emulsifying the aqueous solution ofactive principle thus obtained, with a solution of a polylactide (100%lactide) of average molecular weight 15 000, in methylene chloride. Theemulsification is carried out with a stirrer, with vigorous stirring.Once the first W₁/O emulsion has been formed, it is in turn emulsified,in order to obtain a W₁/O/W₂ emulsion, with an aqueous solution ofpolyvinyl alcohol (0.25%) using a high speed mixer. The double emulsionwhich results therefrom is then stirred gently for several hours inorder to evaporate the organic solvent. The microspheres are thenwashed, recovered by centrifugation and lyophilized. The final load ofthe microspheres with medicinal product is 9 to 11% by weight.

The main differences between the microspheres obtained according to themethod described in the document of the prior art mentioned above andthose obtained according to the method which is the subject of thepresent invention lie in:

-   -   the choice of the polymer. In example 1, the polymer used is a        polylactide (100% lactide) of average molecular weight 15 000.        It is a standard polymer, according to that document, for        prolonged release of        5-OxoPro-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-ProNHEt acetate over a        period of three months;    -   the composition of the aqueous external phase, which consists of        a polyvinyl alcohol (0.25%) in example 1;    -   the system for evaporating the organic solvent. In example 1,        the double emulsion is stirred for several hours.

EXAMPLE 2

According to the method of production which is the subject of thepresent invention, the 5-OxoPro-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-ProNHEtacetate is dissolved in water, and the aqueous solution of activeprinciple is then emulsified, using an ultrasound device, in a solutionof methylene chloride containing 15% of a d,l-lactide-co-glycolidecopolymer, this copolymer having an average molecular weight of 63 000daltons (inherent viscosity of approximately 0.6 dl/g) and a proportionof lactic acid to glycolic acid of 75/25. Once the first emulsion hasbeen formed, it is in turn emulsified with an aqueous solutionconsisting of 0.25% of polysorbate 80, 7% of polyvinylpyrrolidone and 5%of mannitol, with stirring, using a propeller blade stirrer. After thisstep, the double emulsion is passed, under atmospheric conditions, overa continuous evaporating system consisting of a slope of adequatelength, over which the suspension of microspheres flows in a thin layer.Using such an evaporating system, the organic solvent is rapidlyevaporated, resulting in rapid stabilization of the polymer matrix. Themicrospheres are finally recovered by filtration, washed with water, andthen dried under lyophilization. The final load of medicinal product inthe microspheres is 9 to 11% by weight.

EXAMPLE 2a

Microspheres are produced according to the method described in example2, using 5% of sodium chloride in the external aqueous phase as osmoticagent, instead of 5% of mannitol.

EXAMPLE 3

The in vitro release of 5-OxoPro-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-ProNHEtacetate from the microspheres prepared according to examples 1 and 2 isstudied in a phosphate buffer.

25 mg of each formulation in the form of microspheres are suspended in arelease medium made up of 5 ml of phosphate buffer (pH=7.4), and thenstirred (rotation) for 4 days at 37° C. At various times, the amount ofpeptide released was measured by high pressure liquid chromatography(HPLC)

The results of this study are given in table 1. They demonstrate thatthe composition prepared in accordance with the method which is thesubject of the present invention (example 2) exhibits a “burst” releasewhich is much less than the “burst” release of the compositions preparedin accordance with the method of the prior art of example 1. Thus, 4days after the beginning of the study, the microspheres of example 2have released only 3.4% of the total amount of peptide present in themicrospheres, whereas the microspheres of example 1 have released up to22.6% thereof. TABLE 1 % of peptide released Time Example 1 Example 2 1day 13.4% 1.0% 2 days 16.7% 1.4% 4 days 22.6% 3.4%

EXAMPLE 4

The in vivo release of 5-OxoPro-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-ProNHEtacetate from the microspheres prepared according to example 1, 2 and 2ais studied in rats.

For this study., the three type of microspheres (examples 1, 2 and 2a)are suspended in a standard aqueous excipient, before being administeredto rats subcutaneously at a dorsal region shaved beforehand. The doseadministered to each animal is 3.6 mg of microencapsulated5-OxoPro-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-ProNHEt acetate. Some rats aresacrificed three days after administration, whereas others aresacrificed seven days after administration. Once the rats are dead, thesite of injection is then excised, and the microspheres remaining at thesite of injection, along with the contiguous connective tissue, are thenrecovered. The 5-OxoPro-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-ProNHEt acetateremaining is then extracted, and then quantified by high pressure liquidchromatography (HPLC).

The results of this study are given in table 2. It can be seen that,after 3 days, the microspheres of example 1 have already released 35.3%of the total amount of peptide present in the microspheres, whereas themicrospheres of example 2 have released only 20.6%. TABLE 2 % of peptidereleased Time Example 1 Example 2 Example 2a 3 days 35.3% 20.6% 25.9% 5days 41.6% 29.5% —

It may thus be concluded that the results of this in vivo study are inagreement with the results obtained in vitro (example 3) and demonstratethat the formulations prepared in accordance with the method of thepresent invention exhibit a “burst” release which is much lesssubstantial than the “burst” release of the compositions prepared inaccordance with the method of the prior art of example 1.

EXAMPLE 5

In order to complete the preceding results (examples 3 and 4), athorough study of the in vivo release of active principle during thefirst 24 hours following administration of the microspheres was carriedout.

In this trial, the serum content of5-OxoPro-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-ProNHEt acetate is evaluated inrats by liquid chromatography-mass spectrometry-mass spectrometry(LC/MS/MS), after subcutaneous administration of the formulations in theform of microspheres prepared according to examples 1 and 2. Thus, 3.6mg of encapsulated 5-OxoPro-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-ProNHEtacetate are injected into rats and, at various times afteradministration, blood samples are taken in order to estimate the contentof 5-OxoPro-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-ProNHEt acetate in the serum.

The results of this study are given in FIG. 1. Thus, one hour afteradministration of the microspheres of example 1, the concentration ofpeptide in the serum is 107.8 μg/l, whereas, for the microspheres ofexample 2, the concentration of peptide in the serum is 51.8 μg/l. After2, 3, 4, 8 and 24 hours, the microspheres of example 1 exhibit,respectively, serum concentrations of 103.7, 50.1, 30.3, 9.5 and 3.8μg/l, whereas the microspheres of example 2 exhibit, respectively,values of 28.0, 5.5, 4.5, 9.3 and 1.0 μg/l.

Thus, the results of this study are in agreement with the resultsobtained for examples 3 and 4, since it is noted that the microspheresof example 2 release less peptide during the first 24 hours (“burst”effect) than the microspheres of example 1. It may be concluded that,when the microspheres contain the polymer matrix according to thepresent invention, and when they are produced using the method accordingto the present invention, they exhibit a “burst” release which issubstantially less than with the microspheres prepared in accordancewith the method of the prior art, after in vivo administration.

EXAMPLE 6

The complete profile for release of5-OxoPro-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-ProNHEt acetate from themicrospheres prepared according to example 2 is studied in rats. The aimof the study is to demonstrate that, subsequent to the phenomenon of“burst” release (which occurs systematically, but which is relativelylow compared to microspheres described in the prior art), themicrospheres obtained according to the method of the present inventionrelease the peptide continuously over a period of several months.

In a manner similar to example 4, the microspheres are suspended in astandard aqueous vehicle so that they can be administered to ratssubcutaneously at a dorsal region which has been shaved beforehand. Thedose administered to each animal is 3.6 mg of encapsulated5-OxoPro-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-ProNHEt acetate.

At various times over a period of three months, groups of rats aresacrificed, the site of injection is excised, and the microspheresremaining at the site, along with the contiguous connective tissue, arethen recovered. The 5-OxoPro-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-ProNHEtacetate remaining at the site of injection is then extracted, and thenquantified by high pressure liquid chromatography (HPLC).

The results of this study are given in FIG. 2. They thus show that themicrospheres release the peptide continuously over a period of at leastthree months.

EXAMPLE 7

The in vivo biological activity of the5-OxoPro-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-ProNHEt acetatemicro-encapsulated in microspheres prepared according to example 2 andexample 2a is studied here.

5-OxoPro-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-ProNHEt acetate is an analoguewhich is a powerful LH-RH hormone agonist and which stimulatesgonadotrophin secretion by the pituitary gland and steroidogenesis inthe genital organs at acute doses. However, when it is administeredchronically, it produces, paradoxically, agonistic inhibitory effects onpituitary gland gonadotrophin and testicular and ovariansteroidogenesis. Initial administration of5-OxoPro-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-ProNHEt acetate causes an abruptincrease in serum levels of gonadotrophins and of sex hormones, but, inthe case of continuous exposure to the medicinal product, these hormonelevels decrease substantially until they reach levels below the initialbase values, after several days of administration, and this phenomenonpersists for the duration of the treatment.

Consequently, in order to verify that the small “burst” release of theformulations produced according to examples 2 and 2a does not alter itspharmacological activity, 3.6 mg of5-OxoPro-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-ProNHEt acetate microencapsulatedin these microspheres are injected into rats subcutaneously and, atvarious times after administration, blood samples are taken in order toestimate the serum testosterone by radioimmunoassay.

The results of this study are given in FIG. 3. The results thus show anappropriate serum testosterone profile: an abrupt increase during thefirst days, followed by a suppression of the plasma testosteroneconcentration, and they thus demonstrate a correct biological activityof the peptide released from the formulation according to the presentinvention.

The object of examples 8 to 10 is to show the various advantages thatthe microspheres prepared in accordance with the method which is thesubject of the present invention exhibit compared to the microspheresprepared according to the method described in patent FR 2 718 642. Themain differences which exist between the present invention and theinvention described in patent FR 2 718 642 are as follows:

-   -   No release-modulating agent used in the method of the present        invention.    -   Addition of mannitol or of NaCl, as osmotic agent, to the        external aqueous phase in which the first emulsion is        emulsified.    -   Selection of a molecular weight range (40 000 to 80 000) and of        a lactic acid/glycolic acid proportion range (50/50 to 80/20)        for the d,l-lactide-co-glycolide (PLGA) copolymer in the present        invention.

EXAMPLE 8 Effect of the Release-Modulating Agent on the Release In Vitro

Microspheres are prepared according to the method of productiondescribed in example 2, using, as organic phase, a solution of methylenechloride containing 30% of a d,l-lactide-co-glycolide copolymer havingan average molecular weight of 34 000 daltons (inherent viscosity ofapproximately 0.4 dl/g) and a proportion of lactic acid to glycolic acidof 50/50, and also various amounts (examples 8.1, 8.2 and 8.3) of apoly(d,l-lactide) having an average molecular weight of 2000 daltons(PLA 2000).

EXAMPLE 8.1 0% of PLA 2000 EXAMPLE 8.2 2.5% of PLA 2000 EXAMPLE 8.3 5%of PLA 2000

The external aqueous phase is an aqueous solution made up of 0.25% ofpolysorbate 80, 4% of polyvinylpyrrolidone and 5% of mannitol.

The initial in vitro release of5-OxoPro-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-ProNHEt acetate from themicrospheres prepared according to examples 8.1, 8.2 and 8.3 is effectedin a phosphate buffer.

25 mg of each formulation are suspended in a release medium made up of 5ml of phosphate buffer (pH=7.4), and then stirred (rotation) for 7 daysat 37° C. At various times, the amount of peptide released is measuredby high pressure liquid chromatography (HPLC). The results are given intable 3 below. TABLE 3 Time Peptide released in % Days Example 8.1Example 8.2 Example 8.3 2 days 1.1%  1.1%  1.6% 4 days 1.7%  2.7%  2.9%7 days 3.7% 11.8% 18.3%

The results show, for these 3 cases 8.1, 8.2 and 8.3, a very small“burst” release and show that the release-modulating agent (PLA) has aneffect that accelerates the release of the active principle, even at lowconcentrations (2.5%) of PLA, indicating that the use of this agent maynot be suitable for the production of microspheres designed forprolonged release (over a period of three months).

EXAMPLE 9 Effect of the Release-Modulating Agent on the PharmacodynamicProfile and Selection of the Polymer for a Prolonged Release Over ThreeMonths

The effect of the polymer composition of the microspheres on the releaseof the active principle is studied in a pharmacodynamic study carriedout in rats. The microspheres are prepared according to the method ofproduction described in example 2, using various types of polymer:

EXAMPLE 9.1

Mixture of 97.5% of a d,l-lactide-co-glycolide copolymer having anaverage molecular weight of 34 000 daltons and a proportion of lacticacid to glycolic acid of 50/50, and of 2.5% of a poly(d,l-lactide)having an average molecular weight of 2000 daltons (release-modulatingagent).

EXAMPLE 9.2

d,l-lactide-co-glycolide copolymer having an average molecular weight of34 000 daltons and a proportion of lactic acid to glycolic acid of50/50.

EXAMPLE 9.3

d,l-lactide-co-glycolide copolymer having an average molecular weight of63 000 daltons and a proportion of lactic acid to glycolic acid of75/25.

The study of in vivo release of5-OxoPro-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-ProNHEt acetate from themicrospheres is followed through its pharmacological effect ofsuppression of plasma testosterone levels.

The results of this study are given in FIG. 4. The results thus confirmthat the presence of the release-modulating agent accelerates therelease of the active principle, and that the release-modulating agentcannot therefore be used in formulations with very prolonged release(testosterone levels suppressed for 5 weeks).

The results also show that a d-l-lactide-co-glycolide copolymer havingan average molecular weight of 34 000 daltons and a proportion of lacticacid to glycolic acid of 50/50 can maintain the testosterone levelssuppression for 8 weeks, whereas a d,-l-lactide-co-glycolide copolymerhaving an average molecular weight of 63 000 daltons and a proportion oflactic acid to glycolic acid of 75/25 can maintain the castration for atleast three months.

EXAMPLE 10 Effect of the Addition of an Osmotic Agent to the ExternalAqueous Phase Formulation 10.1

Microspheres are prepared according to the method of productiondescribed in example 2, using, as external aqueous phase, a solutionmade up of 0.25% of polysorbate 80 and 4% of polyvinylpyrrolidone.

Formulation 10.2:

Microspheres are prepared according to the method of productiondescribed in example 10.1, adding between 2.5% and 5% of mannitol to theexternal aqueous phase as osmotic agent.

EXAMPLE 10.3

Microspheres are prepared according to the method of productiondescribed in example 10.1, adding between 2.5% and 5% of sodium chlorideto the external aqueous phase as osmotic agent.

The results of the study showing the influence of the osmotic agent inthe external aqueous phase are given in table 4 below: TABLE 4 ExampleOsmotic agent Size μm Content % 10.1 — 59.3 7.7 10.2 2.5% mannitol 50.410.5 10.2   5% mannitol 43.8 10.7 10.3 2.5% NaCl 39.7 10.7 10.3   5%NaCl 32.7 10.4

Thus, the size and the peptide content of the microspheres depend on thepresence of the osmotic agent in the external aqueous phase. Theaddition of mannitol or of NaCl as osmotic agent increases theefficiency of encapsulation, i.e. the percentage of active principlemicroencapsulated (content). The type and the amount of the osmoticagent added can also make it possible to control the size of theparticles. In the context of the present invention, NaCl isadvantageously used as osmotic agent, since it has been demonstratedthat, under identical conditions for stirring and for the viscosity,sodium chloride allows a greater decrease in the size of the particlesthan mannitol, without however producing a decrease in the efficiency ofencapsulation.

EXAMPLE 11 Effect of the Concentration of the Viscosity-Increasing Agent(Viscosity-Modifying Agent) Present in the External Aqueous Phase, onthe Size of the Particles Formulation 11.1

Microspheres are prepared according to the method of productiondescribed in example 2, but without active principle, using, as externalaqueous phase, a solution made up of 0.25% of polysorbate 80, 5% ofmannitol and 6.8% of polyvinylpyrrolidone.

Formulation 11.2

Microspheres are prepared according to the method of productiondescribed in example 2, but without active principle, using, as externalaqueous phase, a solution made up of 0.25% of polysorbate 80, 5% ofmannitol and 8.0% of polyvinylpyrrolidone.

The results of the study showing the influence of theviscosity-modifying agent in the external aqueous phase are given intable 5 below. TABLE 5 Example Viscosity-modifying agent Size μm 11.16.8% polyvinylpyrrolidone 31.3 11.2 8.0% polyvinylpyrrolidone 25.4

Example 10.2, developed with an external aqueous phase made up of 0.25%of polysorbate 80, 5% of mannitol and 4% of polyvinylpyrrolidone, madeit possible to obtain a particle size of 43.8 μm. By increasing theconcentration of viscosity-modifying agent, it can be seen that anotable decrease in the size of the particles is obtained, as shown intable 5 above. Thus, the size of the microspheres depends on thepresence of the viscosity-modifying agent in the external aqueous phase,and on its concentration.

1. A method for preparing a pharmaceutical composition in the form ofmicrospheres with prolonged release of a water-soluble active principle,which comprises the succession of steps below: dissolving the activeprinciple in an appropriate amount of water, emulsifying the aqueoussolution of active principle thus obtained with a solution of ad,l-lactide-co-glycolide matrix copolymer of average molecular weight ofbetween 40 000 and 80 000 and having a lactic acid/glycolic acidproportion of between 50/50 and 80/20, dissolved in a chlorinatedhydrocarbon, resulting in a first microfine and homogeneous emulsion,emulsifying said first emulsion thus obtained, in an external aqueousphase containing a surfactant, a viscosity-increasing agent and anosmotic agent, extracting-evaporating the solvent so as to obtainmicrospheres, which are recovered after filtration, washing and drying.2. The method as claimed in claim 1, wherein the active principle isselected from the group consisting of peptides, proteins, vaccines,antibiotics, antidepressants, analgesics, anti-inflammatories andcytostatics.
 3. The method as claimed in claim 2, wherein the activeprinciple is 5-OxoPro-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-ProNHEt or one ofits salts.
 4. The method as claimed in claim 1, wherein the first stepfor dissolving the active principle in water so as to form an internalaqueous phase is carried out without the addition of any substance whichretains the active principle, nor of agent for stabilizing the emulsion,and without any operation intended to increase the viscosity.
 5. Themethod as claimed in claim 1, wherein the active principle is presentedat a concentration of between 0.01 and 95% by weight relative to thetotal weight of the internal aqueous phase.
 6. The method as claimed inclaim 1, wherein the chlorinated hydrocarbon is methylene chloride. 7.The method as claimed in claim 1, wherein the matrix copolymer ispresent at a concentration of between 5 and 50% by weight relative tothe total weight of the solution consisting of the copolymer dissolvedin the chlorinated hydrocarbon.
 8. The method as claimed in claim 1,wherein the external aqueous phase contains a solution of polysorbate80, of polyvinylpyrrolidone and of mannitol or of sodium chloride,advantageously a solution of polysorbate 80, of polyinylpyrrolidone andof sodium chloride.
 9. The method as claimed in claim 1, wherein thesurfactant is present at a concentration of between 0.1 and 0.5% byweight, the viscosity-increasing agent is present at a concentration ofbetween 1 and 25% by weight and the osmotic agent is present at aconcentration of between 0.1 and 10% by weight, relative to the totalweight of the external aqueous phase.
 10. The method as claimed in claim1, wherein, during the step comprising extraction-evaporation of thesolvent, the solvent is rapidly eliminated at ambient temperature andunder atmospheric pressure, according to a continuous evaporating systemcomprising a slope of adequate length, over which the suspension ofmicrospheres flows in a thin layer.
 11. A microsphere which can beobtained by implementing the method as claimed in claim 1, exhibitingcontinuous release of active principle over a period of more than twomonths.
 12. The microsphere as claimed in claim 11, wherein the activeprinciple is present at a concentration of between 0.5 and 20% byweight, relative to the total weight of the microspheres.
 13. Themicrosphere as claimed in claim 11, wherein said microsphere is lessthan 250 Mm in size.
 14. The microsphere as claimed in claim 11, whereinsaid microsphere is administered parenterally.
 15. The method as claimedin claim 5, wherein the active principle is presented at a concentrationof between 0.5 and 40% by weight, relative to the total weight of theinternal aqueous phase.
 16. A microsphere as claimed in claim 11,exhibiting continuous release of an active principle over a period oftime of at least three months.
 17. The microsphere as claimed in claim12, wherein the active principle is present at a concentration ofbetween 5.0 and 15% by weight relative to the total weight of themicrospheres.
 18. The microspheres as claimed in claim 13, wherein saidmicrosphere is less than 90 μm in size.
 19. The microspheres as claimedin claim 14, wherein said microsphere is administered in the form ofintramuscular injection, subcutaneous injection, intra-arterialinjection, or injection at the site of a tumor.